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Nitric oxide release from rat aortic smooth muscle cells is not attenuated by angiotensin converting enzyme inhibitors
ejp ELSEVIER
European Journal of Pharmacology Molecular PharmacologySection 269 (1994) 319-323
molecular pharmaco!ogy
Nitric oxide release from rat aortic smooth muscle cells is not attenuated by angiotensin converting enzyme inhibitors Uichi Ikeda *, Kazuyuki Shimada Department of Cardiology, Jichi Medical School, Minamikawachi-Machi, Tochigi 329-04, Japan Received 26 May 1994; revised MS received 18 July 1994: accepted 29 July 1994
Abstract
We investigated the effects of angiotensin converting enzyme inhibitors on nitric oxide (NO) synthesis in cultured rat vascular smooth muscle cells. We measured the production of nitrite, a stable metabolitc of NO, and the expression of inducible NO synthase mRNA by vascular smooth muscle cells. Incubation of the culture with interleukin-1/3 (10 ng/ml) for 24 h caused a significant increase in nitrite levels. The basal and interleukin-l/3-induced nitrite production by vascular smooth muscle cells were not affected by the presence of angiotensin converting enzyme inhibitors (0.1 ~ 10 #M), enalaprilat, cilazaprilat or captopril. Unstimulated vascular smooth muscle cells expressed no inducible NO synthase transcripts, whereas incubation with interleukin-1/3 for 24 h induced marked inducible NO synthase mRNA expression. The angiotensin converting enzyme inhibitors, however, had no effects on the interleukin-l/3-induced inducible NO synthasc mRNA expression. These results indicate that angiotensin converting enzyme inhibitors do not attenuate NO synthesis by vascular smooth muscle cells under basal and interleukin-1/3-stimulated conditions.
Keywords: Nitrite; Interleukin-l; Atherosclerosis; Hypertension
1. I n t r o d u c t i o n
Generally, it is accepted that the antihypertensive mechanism of angiotensin converting enzyme inhibitors derives mainly from inhibition of angiotensin II formation. However, studies in experimental animals and in man suggest that they also have vasodilatory activity that is independent of the renin-angiotensin system (Thurston and Sales, 1978; Marks et al., 1980; Man In't Veld et al., 1980; Antonaccio et al., 1981). Several hypothesis have been proposed to explain their undefined vasodilatory activity, but the underlying mechanism has yet to be established unequivocally. There is abundant evidence that angiotensin I1 may be a factor promoting proliferation of fibroblasts, vascular smooth muscle cells and some other cell types (Ikeda et al., 1990; Schelling et al., 1991). Angiotensin converting enzyme inhibitors were shown to have an antiatherogenic effect in Watanabe heritable hyperlipi-
* Corresponding author. Tel.: 81-285-44-2111, Ext. 3557; Fax: 81285-44-5317. 0922-4106/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 9 2 2 - 4 1(16(94)00 130-8
demic rabbits (Chobanian et al., 1990) and to prevent reocclusion after balloon injury in rats (Powell et al., 1989). Recently, Dusting et al. (19931 reported that inhibition of angiotensin converting enzyme with perindopril reduced the initial thickening and partially restored the defective vasodilatation induced by endothelial cell release of nitric oxide (NO) in cuffed arterial segments of rabbit carotid arteries. NO, the best characterized endothelium-derived relaxing factor, is a short-lived free radical. It is synthesized from the amino acid L-arginine in a reaction catalyzed by NO synthase; the terminal nitrogen in the guanidino group is removed from the amino acid and citrulline is formed (Palmer et al., 19881. NO is metabolized rapidly to form nitrite and nitrate. Two forms of NO synthase have been identified: one is expressed constitutively by endothelial cells, brain and myocardium and is dependent on Ca 2+ and calmodulin for activity; the other is inducible and Ca 2+- and calmodulin-independent, and has been identified in endotoxin- and cytokine-treated neutrophils, hepatocytes, endothelial cells and myocardium (Moncada et al., 1991; Dinerman et al., 1993). The inducible NO
320
U. lkeda, K. Shimada / European Journal of Pharmacology - Molecular Pharmacology Section 269 (1994) 319-323
synthase activity is also induced in aortic rings and cultured vascular smooth muscles by cytokines and endotoxins. It has been demonstrated that in vivo balloon injury induces N O synthase activity in rat carotid arteries (Joly et al., 1992). Therefore, N O synthase induction in vascular smooth muscle cells may play a role in vascular contractility and local vascular inflammatory lesions, such as vasculitis, atherosclerosis and arterial injury following balloon angioplasty. Recently, Szab6 et al. (1993) reported that the dihydropyridine calcium channel antagonist nifedipine inhibited the induction of NO synthase by endotoxin in cultured rat vascular smooth muscle cells. However, the effects of angiotensin converting enzyme inhibitors on N O synthesis have not, to our knowledge, been studied. Therefore, in this study, we investigated the effect of three angiotensin converting enzyme inhibitors, enalaprilat, cilazaprilat and captopril, on N O synthesis by vascular smooth muscle cells.
The absorbance at 550 nm was measured and the nitrite concentration was determined by interpolation of a calibration curve of standard sodium nitrite concentrations against absorbance.
2.3. Northern blot analysis
2. Materials and methods
Each required angiotensin converting enzyme inhibitors was added to the culture 60 min before adding interleukin-lfl and incubating for 24 h. The total R N A was extracted from vascular smooth muscle cells using the guanidinium isothiocyanate cesium chloride procedure (Yamamoto et al., 1993), and 15 /~g were subjected to electrophoresis on 1% agarose gels and transferred to nylon filters, which were hybridized with a random primed, 32p-labeled rat inducible NO synthase (Koide et al., 1993) and 18S r R N A c D N A probes for 24 h, washed twice with an aqueous solution of 150 mM NaC1, 15 mM sodium citrate, and 0.1% sodium dodecyl sulfate at 65°C and then Kodak XAR-5 film was exposed to the filters for 1 ~ 2 days at - 7 0 ° C using one intensifying screen.
2.1. Culture of cells
2.4. Statistical analysis
Primary cultures of vascular smooth muscle cells were obtained from the media of thoracic aortae of Sprague Dawley rats (200 ~ 250 g), as described previously (Ikeda et al., 1991). The cells were grown in Dulbecco's Minimum Essential Medium ( D M E M ) supplemented with 10% fetal bovine serum, 100 U / m l penicillin and 100 / ~ g / m l streptomycin. The cultures were harvested twice a week treated with 0.125% trypsin and passaged at a 1 : 3 ratio in 100-mm culture dishes (Falcon, N J). The typical growth experiment was performed using cultured cells at passage levels of 5 ~ 10. Cells (1.5 × 104/well) were plated in 24-well dishes (Falcon, N J) in D M E M , supplemented as described above, and allowed to grow to subconfluence for 24 ~ 48 h after which, they were preincubated in 0.5% fetal bovine serum-containing D M E M supplemented with insulin (5 ~zg/ml) and transferrin (5 g g / ml) for 24 h, and used for the experiments described below.
The data are expressed as means _+ S.E. For comparisons between multiple groups, we determined the probability that differences existed between the means of the groups by analysis of variances using the least significant difference for multiple comparisons. Differences at P values of <0.05 were considered to be statistically significant.
2.5. Materials H u m a n recombinant interleukin-lfl was obtained from Genzyme (Boston, MA), the rat inducible N O synthase c D N A probe was a gift from Dr. Y. Kawahara (Kobe University School of Medicine), NG-mono methyl-L-arginine, enalaprilat and captopril were obtained from Sigma (MO) and cilazaprilat was a gift from Roche Japan (Tokyo). All the other chemicals used were of the highest grade commercially available.
2.2. Measurement of nitrite
3. Results
The N O production by the cultured cells was determined by measuring the nitrite contents of the culture media. The vascular smooth muscle cells were incubated in 0.5% fetal bovine serum-containing D M E M at 37°C, and the nitrite content of each culture medium was determined by mixing 500 p.1 medium with an equal volume of Griess reagent (1 part 0.1% naphthylethylenediamine dihydrochloride to 1 part 1% sulfanilamide in 5% phosphoric acid) (Green et al., 1982).
First, we investigated the effect of interleukin-1 fl on nitrite production by vascular smooth muscle cells. As shown in Fig. 1, interleukin-lfl (10 n g / m l ) stimulated nitrite production by vascular smooth muscle cells in a time-dependent manner. In the presence of the N O synthesis inhibitor N%monomethyl-L-arginine (1 mM), no interleukin-1/3-induced nitrite production was observed. Next, we investigated the effects of three an-
U. Ikeda, K. Shimada / European Journal of Pharmacology - Molecular Pharmacology Section 269 (1994) 319-323 120.
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Time (hr) Fig. 1. Effect of interleukin-1/3 on nitrite production by cultured rat vascular smooth muscle cells. Cells were exposed to interleukin-l/3 (10 n g / m l ) in the presence ( o ) or absence (e) of NC'-monomethyl L-arginine (1 mM) for the indicated periods. Data are m e a n s + S . E . of four samples; * P <0.01 compared with control cells ([3) not exposed to interleukin-1/3.
giotensin converting enzyme inhibitors, enalaprilat, cilazaprilat and captopril, on the interleukin-1/3-induced nitrite synthesis by vascular smooth muscle cells. Adding each of these inhibitors alone to the cultures and incubating them for 24 h had no effects on the basal production of nitrite by vascular smooth muscle cells (data not shown). Incubation with interleukin-1/3 (10 n g / m l ) for 24 h induced a significant increase in nitrite levels, but the interleukin-1/3-raised levels were not affected by the presence of 0.1 ~ 10 tzM enalaprilat, cilazaprilat or captopril (Fig. 2). 120
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IL-1 [3 (ng/ml) Fig. 3. Effect of enalaprilat, cilazaprilat or captopril on various concentrations of interleukin-1/3-induced nitrite production by vascular smooth muscle cells. Cells were exposed to various concentrations of interleukin-l/3 (0.1 ~ 10 n g / m l ) for 24 h with 1 ~zM enalaprilat, cilazaprilat or captopril, which was added 60 min before interleukin1/3. Data are means+_ S.E. of four experiments.
We further confirmed the ineffectiveness of these angiotensin converting enzyme inhibitors on the interleukin-1/3-induced nitrite synthesis using various concentrations of interleukin-1/3 (0.1 ~ 10 n g / m l ) . As shown in Fig. 3, 1 /xM enalaprilat, cilazaprilat or captopril showed no significant effect on nitrite production by vascular smooth muscle cells induced by various concentrations of interleukin-1/3. Finally, we studied inducible NO synthase m R N A expression by vascular smooth muscle cells. Unstimulated vascular smooth muscle cells expressed almost no inducible NO synthase transcripts, whereas incubation with interleukin-1/3 (10 n g / m l ) for 24 h resulted in marked induction of inducible NO synthase m R N A expression. Addition of each of three angiotensin converting enzyme inhibitors (1 /xM) described above did not affect the interleukin-1/3-induced inducible NO synthase m R N A expression by vascular smooth muscle cells (Fig. 4).
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Fig. 2. Effect of various concentrations of angiotensin converting enzyme inhibitors on interleukin-1/3-induced nitrite production by vascular smooth muscle cells. Cells were exposed to interleukin-1/3 (10 n g / m l ) for 24 h with various concentrations (0.1 ~ 10 txM) of enalaprilat, cilazaprilat or captopril, which was added 60 min before interleukin-1/3. Data are means + S.E. of four experiments.
In this study, we investigated whether angiotensin converting enzyme inhibitors affected NO synthesis by cultured rat vascular smooth muscle cells. Neither enalaprilat, cilazaprilat nor captopril had any significant effect on NO synthesis by vascular smooth muscle cells under basal and interleukin-1/3-stimulated conditions. The inducible NO synthase activity is induced in the blood vessel wall and in cultured vascular smooth muscle cells by endotoxin and cytokines (Dinerman et al.,
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Fig. 4. Expression of inducible NO synthase mRNA by vascular smooth muscle cells stimulated by interleukin-1/3 in the presence of angiotensin converting enzyme inhibitors. Cells were incubated with interleukin-l/3 (10 ng/ml) for 24 h with 1 ~M enalaprilat, cilazaprilat or captopril, which were added to the culture medium 60 min before interleukin-l/3. Three independent experiments yielded identical results.
1993). NO thus produced has been implicated not only in sepsis-related systemic hypotension but also in the pathogenesis of atherosclerosis and reocclusion after balloon injury (Garg and Hassid, 1989; Joly et al., 1992; M c N a m a r a et al., 1993). Hypercholesterolemia and atherosclerosis in laboratory animals and in man are associated with impaired endothelium-mediated vasodilatation in vitro and in vivo (F6rstermann et al., 1988; Minor et al., 1990). Studies on arterial segments from hyperchotesterolemic rabbits indicated that breakdown of N O is increased compared with normal rabbits, probably due to enhanced formation of superoxide anions ( . 0 2) (Minor et al., 1990). A recent report by O h a r a et al. (1992) demonstrated elevated -O 2 formation mainly in the intima of atherosclerotic arteries, thus lending further support to the hypothesis that the impaired vasodilatation associated with atherosclerosis is due to more rapid oxidation of N O than that in normal vascular tissue. Recent studies demonstrated that in vivo balloon injury induces N O synthase activity in rat carotid arteries, even in the absence of the endothelium (Joly et al., 1992), and that the angiotensin converting enzyme inhibitors cilazapril and captopril can reduce the myointimal thickening that occurs in response to balloon catheter injury of the rat
-
Molecular Pharmacology Section 269 (1994) 319-323
carotid artery (Powell, et al., 1989; Clozel et al., 1993). These interesting findings support the concept that NO and angiotensin converting enzyme inhibitors act on cellular proliferation in this in vivo vascular injury model, although how the latter finding relates to the attenuating effects of angiotensin converting enzyme inhibitors on N O production is uncertain. In this study, we observed no significant effects of angiotensin converting enzyme inhibitors on N O production by vascular smooth muscle cells in vitro. However, we cannot rule out the possibility that angiotensin converting enzyme inhibitors may have effects on production a n d / o r stabilization of NO in atherosclerotic vascular tissue in vivo. The results of a recent experimental study suggested that the effects of angiotensin converting enzyme inhibition on neointimal formation may not be due to decreased angiotensin lI formation but to accumulation of bradykinin, which is degraded by angiotensin converting enzyme, in the vascular tissues (Farhy et al., 1992). Several investigators (Miigge et al., 1991; Gr:,ife et al., 1993; Emori et al., 1993) showed that bradykinin stimulated NO production by human and bovine endothelial cells via constitutive NO synthase activation, but at concentrations of 0.1 ~ 10/xM, we found it had no effect on inducible N O synthase induction by rat vascular smooth muscle cells (data not shown). Reduced endothelium-mediated vasoditatation also has been demonstrated in arteries from hypertensive animals and impaired responses to endothelium-derived relaxing factor have been observed in hypertensive patients compared with normotensive subjects. The generally accepted major antihypertensive mechanism of action of angiotensin converting enzyme inhibitors is inhibition of angiotensin II formation. However, studies in experimental animals and in man suggested that they also have vasodilatory activity that is independent of the renin-angiotensin system (Thurston and Sales, 1978; Marks et al., 1980; Man In 't Veld et al., 1980; Antonaccio et al., 1981). Goldschmidt et al. (1991) demonstrated that captopril possesses an endotheliumdependent vasodilator component that is related neither to angiotensin converting enzyme inhibition nor to a prostaglandin-mediated process and that the action is due to its sulfhydryl (SH) group and its ability to scavenge . 0 2 and thereby stabilize NO. If NO synthase induction is stimulated by angiotensin converting enzyme inhibitors in vascular smooth muscle cells, then part of the antihypertensive effect may be attributable to increased NO synthesis. However, we observed no significant effects of angiotensin converting enzyme inhibitors on inducible N O synthase induction in vascular smooth muscle cells under basal and interleukin1/3-stimulated conditions. In summary, we showed that angiotensin converting enzyme inhibitors did not affect NO synthesis by vascu-
U. lkeda, K. Shimada / European Journal of Pharmacology - Molecular Pharmacology Section 269 (19941 319-323
lar smooth muscle cells in vitro. However, we do not know what effects angiotensin converting enzyme inhibitors may have on NO synthesis by vascular endothelial cells or vascular tissues under various pathophysiological conditions and further studies are needed to clarify these points.
Acknowledgments We wish to thank Toshiko Kanbe for her technical assistance. This study was supported by the Ministry of Education, Culture and Science of Japan (#5670632, 5770477).
References Antonaccio, M.J., B. Rubin and D. Kotler, 1981, Effects of captopril on vascular reactivity of SHR in vivo and in vitro, Hypertension 3, 11-211. Chobanian, A.V., C. Haudenschild, C. Nickerson and R. Drago, 1990, Antiatherogenic effect of captopril in the Watanabe heritable hyperlipidemic rabbit, Hypertension 15,327. Clozel, J.P., K.M. Rita, M.D. Nat, S. Roux, W. Fischli and H.R. Baumgartner, 1993, Influence of the status of the renin-angiotensin system on the effect of cilazapril on neointima formation after vascular injury in rats, Circulation 88, 1222. Dinerman, J.L., C.J. Lowenstein and S.H. Snyder, 1993, Molecular mechanisms of nitric oxide regulation, Circ. Res. 73, 217. Dusting, C.F., M. Makdissi and H. Hickey, 1993, Perindopril reduces neo-intima development and partially restores endothelial nitric oxide function in rabbit carotid arteries, Current Advances in ACE Inhibition, eds. G.A. Macgregor and P.S. Sever (Churchill Livingstone, London) p. 142. Emori, T., Hirata, Y., Kanno, K. and F. Marumo, 1993, Bradykinin stimulates endothelial nitric oxide (NO) production by calcium/ calmodulin-dependent NO synthase, Hypertens. Res. 16, 131. Farhy, R.D., K.L. Ho, O.A. Carretero and A.G. Scicli, 1992, Kinins mediate the antiproliferative effect of ramipril in rat carotid artery, Biochem. Biophys. Res. Commun. 182, 283. F6rstermann, U., A. Mfigge, U. Alheid, A. Haverich and J.C. Fr61ich, 1988, Selective attenuation of endothelium-mediated vasodilatation in atherosclerotic human coronary arteries, Circ. Res. 62, 185. Garg, U.C. and A. Hassid, 1989, Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells, J. Clin. Invest. 83, 1774. Goldschmidt, J.E. and R.J. Tallarida, 1991, Pharmacological evidence that captopril possesses an endothelium-mediated component of vasodilatation: Effect of sulfhydryl groups on endothelium-derived relaxing factor, J. Pharmacol. Exp. Ther. 257, 1136. Grille, M., C. Bossaller, G. Kristof, W. Auch-Schwelk, C.R. Baumgarten, A. Hildebrandt and E. Fleck, 1993, Effect of angiotensinconverting-enzyme inhibition on bradykinin metabolism by vascular endothelial cells, Am. J. Physiol. 265, H1493.
323
Green, L.C., D.A. Wagner, J. Glogowski, P.L. Skiper, J.S. Wishnok and S.R. Tannenbaum, 1982, Analysis of nitrate, nitrite and [tSN]nitrate in biological fluids, Anal. Biochem. 126, 131. Ikeda, U., M. Ikeda, T. Oohara, S. Kano and T. Yaginuma, 19911, Mitogenic action of interleukin-la on vascular smooth muscle cells mediated by PDGF, Atherosclerosis 84, 183. Ikeda, U., M. Ikeda, T. Oohara, A. Oguchi, T. Kamitani, Y. Tsuruya and S. Kano, 1991, lnterleukin 6 stimulates growth of vascular smooth muscle cells in a PDGF-dependent manner, Am. J. Physiol. 260, H1713. Joly, G.A., V.B. Schini and P.M. Vanhoutte, 1992, Balloon injury and interleukin-1/3 induce nitric oxide synthase activity in rat carotid cateries, Circ. Res. 71,331. Koide, M., Y. Kawahara, T. Tsuda and M. Yokoyama, 1993, Cytokine-induced expression of an inducible type of nitric oxide synthase gene in cultured vascular smooth muscle cells, FEBS Lett. 318, 213. Man In't Veld, A.J., I.M. Schichit, F.M. Derkx, J.H.B. Debruyn and M.A.D.H. Schalekamp, 1980, Effect of captopril on blood pressure in anephric subjects, Br. Med. J. 1,288. Marks, E.S., R.F. Bing, H. Thurston and J.D. Swales, 1980, Vasodepressor property of the converting enzyme inhibitor captopril (SQ 14,225): the role of factors other than renin-angiotensin blockade in the rat, Clin. Sci. 58, 1. McNamara, D.B., B. Bedi, A. Harmeat, H. Aurora, L. Tcna, L.J. lgnarro, P.J. Kadowitz and D.L. Akers, 1993, ~-Arginine inhibits balloon catheter-induced intimal hyperplasia, Biochem. Biophys. Res. Commun. 193, 291. Minor, R.L., P.R. Myers, R. Guerra, J.N. Bates and D.G. Harrison, 1990, Diet-induced atherosclerosis increases the release of nitrogen oxides from rabbit aorta, J. Clin. Invest. 86, 2109. Moncada, S., R.M.J. Palmer and E.A. Higgs, 1991, Nitric oxide: physiology, pathophysiology and pharmacology, Pharmacol. Rev. 43, 109. Miigge, A., T. Peterson and D.G. Harrison, 1991, Release of nitrogen oxides from cultured bovine aortic endothelial cells is not impaired by calcium channel antagonists, Circulation 83, 1404. Ohara, Y., T.E. Peterson and D.G. Harrison, 1992, Hypercholesterolemia increases superoxide anion production by the endothelium [abstr.], Circulation 86, 1-222. Palmer, R.M.J., D.S. Ashton and S. Moncada, 1988, Vascular endothelial cells synthesize nitric oxide from L-arginine, Nature 333, 664. Powell, S., J.P. Clozel, R. Muller, H. Kunh, F. Hefti, M. Hosang and H.R. Baumgartner, 1989, Inhibitors of angiotensin-converting enzyme prevent myointimal proliferation after vascular injury, Science 245, 186. Schelling, P., H. Fischer and D. Ganten, 1991, Angiotensin and cell growth: A link to cardiovascular hypertrophy, J. Hypertens. 9, 3. Szab6, C., J.A. Mitchell, S.S. Gross, C. Thiemermann and J.R. Vane, 1993, Nifedipine inhibits the induction of nitric oxide synthase by bacterial lipopolysaccharide, J. Pharmacol. Exp. Ther. 265, 674. Thurston, H. and J.D. Sales, 1978, Converting enzyme inhibitor and saralasin infusion in rats: Evidence for an additional vasodepressor property of converting-enzyme inhibitor, Circ. Res. 42, 588. Yamamoto, K., U. Ikeda, Y. Seino, Y. Tsuruya, A. Oguchi, K. Okada, S. lshikawa, T. Saito, K. Kawakami, Y. Hara and K. Shimada, 1993, Regulation of Na,K-ATPase gene expression by sodium ions in cultured neonatal rat cardiocytes, J. Clin. Invest. 92, 1889.
European Journal of Pharmacology Molecular PharmacologySection 269 (1994) 319-323
molecular pharmaco!ogy
Nitric oxide release from rat aortic smooth muscle cells is not attenuated by angiotensin converting enzyme inhibitors Uichi Ikeda *, Kazuyuki Shimada Department of Cardiology, Jichi Medical School, Minamikawachi-Machi, Tochigi 329-04, Japan Received 26 May 1994; revised MS received 18 July 1994: accepted 29 July 1994
Abstract
We investigated the effects of angiotensin converting enzyme inhibitors on nitric oxide (NO) synthesis in cultured rat vascular smooth muscle cells. We measured the production of nitrite, a stable metabolitc of NO, and the expression of inducible NO synthase mRNA by vascular smooth muscle cells. Incubation of the culture with interleukin-1/3 (10 ng/ml) for 24 h caused a significant increase in nitrite levels. The basal and interleukin-l/3-induced nitrite production by vascular smooth muscle cells were not affected by the presence of angiotensin converting enzyme inhibitors (0.1 ~ 10 #M), enalaprilat, cilazaprilat or captopril. Unstimulated vascular smooth muscle cells expressed no inducible NO synthase transcripts, whereas incubation with interleukin-1/3 for 24 h induced marked inducible NO synthase mRNA expression. The angiotensin converting enzyme inhibitors, however, had no effects on the interleukin-l/3-induced inducible NO synthasc mRNA expression. These results indicate that angiotensin converting enzyme inhibitors do not attenuate NO synthesis by vascular smooth muscle cells under basal and interleukin-1/3-stimulated conditions.
Keywords: Nitrite; Interleukin-l; Atherosclerosis; Hypertension
1. I n t r o d u c t i o n
Generally, it is accepted that the antihypertensive mechanism of angiotensin converting enzyme inhibitors derives mainly from inhibition of angiotensin II formation. However, studies in experimental animals and in man suggest that they also have vasodilatory activity that is independent of the renin-angiotensin system (Thurston and Sales, 1978; Marks et al., 1980; Man In't Veld et al., 1980; Antonaccio et al., 1981). Several hypothesis have been proposed to explain their undefined vasodilatory activity, but the underlying mechanism has yet to be established unequivocally. There is abundant evidence that angiotensin I1 may be a factor promoting proliferation of fibroblasts, vascular smooth muscle cells and some other cell types (Ikeda et al., 1990; Schelling et al., 1991). Angiotensin converting enzyme inhibitors were shown to have an antiatherogenic effect in Watanabe heritable hyperlipi-
* Corresponding author. Tel.: 81-285-44-2111, Ext. 3557; Fax: 81285-44-5317. 0922-4106/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 9 2 2 - 4 1(16(94)00 130-8
demic rabbits (Chobanian et al., 1990) and to prevent reocclusion after balloon injury in rats (Powell et al., 1989). Recently, Dusting et al. (19931 reported that inhibition of angiotensin converting enzyme with perindopril reduced the initial thickening and partially restored the defective vasodilatation induced by endothelial cell release of nitric oxide (NO) in cuffed arterial segments of rabbit carotid arteries. NO, the best characterized endothelium-derived relaxing factor, is a short-lived free radical. It is synthesized from the amino acid L-arginine in a reaction catalyzed by NO synthase; the terminal nitrogen in the guanidino group is removed from the amino acid and citrulline is formed (Palmer et al., 19881. NO is metabolized rapidly to form nitrite and nitrate. Two forms of NO synthase have been identified: one is expressed constitutively by endothelial cells, brain and myocardium and is dependent on Ca 2+ and calmodulin for activity; the other is inducible and Ca 2+- and calmodulin-independent, and has been identified in endotoxin- and cytokine-treated neutrophils, hepatocytes, endothelial cells and myocardium (Moncada et al., 1991; Dinerman et al., 1993). The inducible NO
320
U. lkeda, K. Shimada / European Journal of Pharmacology - Molecular Pharmacology Section 269 (1994) 319-323
synthase activity is also induced in aortic rings and cultured vascular smooth muscles by cytokines and endotoxins. It has been demonstrated that in vivo balloon injury induces N O synthase activity in rat carotid arteries (Joly et al., 1992). Therefore, N O synthase induction in vascular smooth muscle cells may play a role in vascular contractility and local vascular inflammatory lesions, such as vasculitis, atherosclerosis and arterial injury following balloon angioplasty. Recently, Szab6 et al. (1993) reported that the dihydropyridine calcium channel antagonist nifedipine inhibited the induction of NO synthase by endotoxin in cultured rat vascular smooth muscle cells. However, the effects of angiotensin converting enzyme inhibitors on N O synthesis have not, to our knowledge, been studied. Therefore, in this study, we investigated the effect of three angiotensin converting enzyme inhibitors, enalaprilat, cilazaprilat and captopril, on N O synthesis by vascular smooth muscle cells.
The absorbance at 550 nm was measured and the nitrite concentration was determined by interpolation of a calibration curve of standard sodium nitrite concentrations against absorbance.
2.3. Northern blot analysis
2. Materials and methods
Each required angiotensin converting enzyme inhibitors was added to the culture 60 min before adding interleukin-lfl and incubating for 24 h. The total R N A was extracted from vascular smooth muscle cells using the guanidinium isothiocyanate cesium chloride procedure (Yamamoto et al., 1993), and 15 /~g were subjected to electrophoresis on 1% agarose gels and transferred to nylon filters, which were hybridized with a random primed, 32p-labeled rat inducible NO synthase (Koide et al., 1993) and 18S r R N A c D N A probes for 24 h, washed twice with an aqueous solution of 150 mM NaC1, 15 mM sodium citrate, and 0.1% sodium dodecyl sulfate at 65°C and then Kodak XAR-5 film was exposed to the filters for 1 ~ 2 days at - 7 0 ° C using one intensifying screen.
2.1. Culture of cells
2.4. Statistical analysis
Primary cultures of vascular smooth muscle cells were obtained from the media of thoracic aortae of Sprague Dawley rats (200 ~ 250 g), as described previously (Ikeda et al., 1991). The cells were grown in Dulbecco's Minimum Essential Medium ( D M E M ) supplemented with 10% fetal bovine serum, 100 U / m l penicillin and 100 / ~ g / m l streptomycin. The cultures were harvested twice a week treated with 0.125% trypsin and passaged at a 1 : 3 ratio in 100-mm culture dishes (Falcon, N J). The typical growth experiment was performed using cultured cells at passage levels of 5 ~ 10. Cells (1.5 × 104/well) were plated in 24-well dishes (Falcon, N J) in D M E M , supplemented as described above, and allowed to grow to subconfluence for 24 ~ 48 h after which, they were preincubated in 0.5% fetal bovine serum-containing D M E M supplemented with insulin (5 ~zg/ml) and transferrin (5 g g / ml) for 24 h, and used for the experiments described below.
The data are expressed as means _+ S.E. For comparisons between multiple groups, we determined the probability that differences existed between the means of the groups by analysis of variances using the least significant difference for multiple comparisons. Differences at P values of <0.05 were considered to be statistically significant.
2.5. Materials H u m a n recombinant interleukin-lfl was obtained from Genzyme (Boston, MA), the rat inducible N O synthase c D N A probe was a gift from Dr. Y. Kawahara (Kobe University School of Medicine), NG-mono methyl-L-arginine, enalaprilat and captopril were obtained from Sigma (MO) and cilazaprilat was a gift from Roche Japan (Tokyo). All the other chemicals used were of the highest grade commercially available.
2.2. Measurement of nitrite
3. Results
The N O production by the cultured cells was determined by measuring the nitrite contents of the culture media. The vascular smooth muscle cells were incubated in 0.5% fetal bovine serum-containing D M E M at 37°C, and the nitrite content of each culture medium was determined by mixing 500 p.1 medium with an equal volume of Griess reagent (1 part 0.1% naphthylethylenediamine dihydrochloride to 1 part 1% sulfanilamide in 5% phosphoric acid) (Green et al., 1982).
First, we investigated the effect of interleukin-1 fl on nitrite production by vascular smooth muscle cells. As shown in Fig. 1, interleukin-lfl (10 n g / m l ) stimulated nitrite production by vascular smooth muscle cells in a time-dependent manner. In the presence of the N O synthesis inhibitor N%monomethyl-L-arginine (1 mM), no interleukin-1/3-induced nitrite production was observed. Next, we investigated the effects of three an-
U. Ikeda, K. Shimada / European Journal of Pharmacology - Molecular Pharmacology Section 269 (1994) 319-323 120.
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giotensin converting enzyme inhibitors, enalaprilat, cilazaprilat and captopril, on the interleukin-1/3-induced nitrite synthesis by vascular smooth muscle cells. Adding each of these inhibitors alone to the cultures and incubating them for 24 h had no effects on the basal production of nitrite by vascular smooth muscle cells (data not shown). Incubation with interleukin-1/3 (10 n g / m l ) for 24 h induced a significant increase in nitrite levels, but the interleukin-1/3-raised levels were not affected by the presence of 0.1 ~ 10 tzM enalaprilat, cilazaprilat or captopril (Fig. 2). 120
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We further confirmed the ineffectiveness of these angiotensin converting enzyme inhibitors on the interleukin-1/3-induced nitrite synthesis using various concentrations of interleukin-1/3 (0.1 ~ 10 n g / m l ) . As shown in Fig. 3, 1 /xM enalaprilat, cilazaprilat or captopril showed no significant effect on nitrite production by vascular smooth muscle cells induced by various concentrations of interleukin-1/3. Finally, we studied inducible NO synthase m R N A expression by vascular smooth muscle cells. Unstimulated vascular smooth muscle cells expressed almost no inducible NO synthase transcripts, whereas incubation with interleukin-1/3 (10 n g / m l ) for 24 h resulted in marked induction of inducible NO synthase m R N A expression. Addition of each of three angiotensin converting enzyme inhibitors (1 /xM) described above did not affect the interleukin-1/3-induced inducible NO synthase m R N A expression by vascular smooth muscle cells (Fig. 4).
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Fig. 2. Effect of various concentrations of angiotensin converting enzyme inhibitors on interleukin-1/3-induced nitrite production by vascular smooth muscle cells. Cells were exposed to interleukin-1/3 (10 n g / m l ) for 24 h with various concentrations (0.1 ~ 10 txM) of enalaprilat, cilazaprilat or captopril, which was added 60 min before interleukin-1/3. Data are means + S.E. of four experiments.
In this study, we investigated whether angiotensin converting enzyme inhibitors affected NO synthesis by cultured rat vascular smooth muscle cells. Neither enalaprilat, cilazaprilat nor captopril had any significant effect on NO synthesis by vascular smooth muscle cells under basal and interleukin-1/3-stimulated conditions. The inducible NO synthase activity is induced in the blood vessel wall and in cultured vascular smooth muscle cells by endotoxin and cytokines (Dinerman et al.,
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Fig. 4. Expression of inducible NO synthase mRNA by vascular smooth muscle cells stimulated by interleukin-1/3 in the presence of angiotensin converting enzyme inhibitors. Cells were incubated with interleukin-l/3 (10 ng/ml) for 24 h with 1 ~M enalaprilat, cilazaprilat or captopril, which were added to the culture medium 60 min before interleukin-l/3. Three independent experiments yielded identical results.
1993). NO thus produced has been implicated not only in sepsis-related systemic hypotension but also in the pathogenesis of atherosclerosis and reocclusion after balloon injury (Garg and Hassid, 1989; Joly et al., 1992; M c N a m a r a et al., 1993). Hypercholesterolemia and atherosclerosis in laboratory animals and in man are associated with impaired endothelium-mediated vasodilatation in vitro and in vivo (F6rstermann et al., 1988; Minor et al., 1990). Studies on arterial segments from hyperchotesterolemic rabbits indicated that breakdown of N O is increased compared with normal rabbits, probably due to enhanced formation of superoxide anions ( . 0 2) (Minor et al., 1990). A recent report by O h a r a et al. (1992) demonstrated elevated -O 2 formation mainly in the intima of atherosclerotic arteries, thus lending further support to the hypothesis that the impaired vasodilatation associated with atherosclerosis is due to more rapid oxidation of N O than that in normal vascular tissue. Recent studies demonstrated that in vivo balloon injury induces N O synthase activity in rat carotid arteries, even in the absence of the endothelium (Joly et al., 1992), and that the angiotensin converting enzyme inhibitors cilazapril and captopril can reduce the myointimal thickening that occurs in response to balloon catheter injury of the rat
-
Molecular Pharmacology Section 269 (1994) 319-323
carotid artery (Powell, et al., 1989; Clozel et al., 1993). These interesting findings support the concept that NO and angiotensin converting enzyme inhibitors act on cellular proliferation in this in vivo vascular injury model, although how the latter finding relates to the attenuating effects of angiotensin converting enzyme inhibitors on N O production is uncertain. In this study, we observed no significant effects of angiotensin converting enzyme inhibitors on N O production by vascular smooth muscle cells in vitro. However, we cannot rule out the possibility that angiotensin converting enzyme inhibitors may have effects on production a n d / o r stabilization of NO in atherosclerotic vascular tissue in vivo. The results of a recent experimental study suggested that the effects of angiotensin converting enzyme inhibition on neointimal formation may not be due to decreased angiotensin lI formation but to accumulation of bradykinin, which is degraded by angiotensin converting enzyme, in the vascular tissues (Farhy et al., 1992). Several investigators (Miigge et al., 1991; Gr:,ife et al., 1993; Emori et al., 1993) showed that bradykinin stimulated NO production by human and bovine endothelial cells via constitutive NO synthase activation, but at concentrations of 0.1 ~ 10/xM, we found it had no effect on inducible N O synthase induction by rat vascular smooth muscle cells (data not shown). Reduced endothelium-mediated vasoditatation also has been demonstrated in arteries from hypertensive animals and impaired responses to endothelium-derived relaxing factor have been observed in hypertensive patients compared with normotensive subjects. The generally accepted major antihypertensive mechanism of action of angiotensin converting enzyme inhibitors is inhibition of angiotensin II formation. However, studies in experimental animals and in man suggested that they also have vasodilatory activity that is independent of the renin-angiotensin system (Thurston and Sales, 1978; Marks et al., 1980; Man In 't Veld et al., 1980; Antonaccio et al., 1981). Goldschmidt et al. (1991) demonstrated that captopril possesses an endotheliumdependent vasodilator component that is related neither to angiotensin converting enzyme inhibition nor to a prostaglandin-mediated process and that the action is due to its sulfhydryl (SH) group and its ability to scavenge . 0 2 and thereby stabilize NO. If NO synthase induction is stimulated by angiotensin converting enzyme inhibitors in vascular smooth muscle cells, then part of the antihypertensive effect may be attributable to increased NO synthesis. However, we observed no significant effects of angiotensin converting enzyme inhibitors on inducible N O synthase induction in vascular smooth muscle cells under basal and interleukin1/3-stimulated conditions. In summary, we showed that angiotensin converting enzyme inhibitors did not affect NO synthesis by vascu-
U. lkeda, K. Shimada / European Journal of Pharmacology - Molecular Pharmacology Section 269 (19941 319-323
lar smooth muscle cells in vitro. However, we do not know what effects angiotensin converting enzyme inhibitors may have on NO synthesis by vascular endothelial cells or vascular tissues under various pathophysiological conditions and further studies are needed to clarify these points.
Acknowledgments We wish to thank Toshiko Kanbe for her technical assistance. This study was supported by the Ministry of Education, Culture and Science of Japan (#5670632, 5770477).
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